Data setup and objectives

Cohort demographics

Stool data

level HDC MAH SAH p
n 20 8 21
age (mean (SD)) 46.39 (13.63) 54.33 (7.09) 43.21 (11.36) 0.089
GENDER (%) F 8 (40.0) 1 (12.5) 11 ( 52.4) 0.148
M 12 (60.0) 7 (87.5) 10 ( 47.6)
BMI (mean (SD)) 29.56 (7.51) 26.92 (2.60) 28.35 (6.09) 0.673
race (%) Black or African American 1 ( 5.0) 1 (12.5) 0 ( 0.0) 0.421
More than one race 1 ( 5.0) 0 ( 0.0) 0 ( 0.0)
White 18 (90.0) 7 (87.5) 21 (100.0)
HEMOGLOBIN (mean (SD)) 13.21 (1.43) 9.84 (1.82) 10.36 (1.90) <0.001
WBC (mean (SD)) 6.75 (2.69) 13.74 (8.70) 16.56 (12.83) 0.008
PLATELET (mean (SD)) 265.89 (52.83) 207.60 (175.51) 155.00 (88.64) 0.002
CREATININE (mean (SD)) 0.89 (0.35) 0.67 (0.31) 1.09 (0.71) 0.257
AST (mean (SD)) 25.00 (8.27) 158.20 (91.16) 114.50 (44.97) <0.001
ALT (mean (SD)) 25.11 (12.36) 63.20 (42.86) 40.40 (21.07) 0.002
TOTAL_BILIRUBIN (mean (SD)) 0.44 (0.27) 7.06 (3.72) 19.57 (11.36) <0.001
ALBUMIN (mean (SD)) 4.06 (0.52) 2.66 (0.15) 2.82 (0.52) <0.001
INR (mean (SD)) 1.00 (0.15) 1.37 (0.20) 2.07 (0.40) <0.001
MELD_SCORE (mean (SD)) 7.37 (1.74) 14.62 (3.85) 26.81 (5.32) <0.001
CHILDPUGH_SCORE (mean (SD)) 5.21 (0.43) 9.00 (0.71) 10.45 (1.64) <0.001
DF_SCORE (mean (SD)) 12.54 (8.79) 35.95 (7.00) 81.33 (31.75) <0.001
TLFB_AVG_drinksDD (mean (SD)) 17.32 (13.42) 16.40 (13.71) 12.44 (9.13) 0.434

Association between demographic variables and bile acids

Stool metabolites univariate meld prediction

Plasma data

level HC HDC MAH SAH p
n 20 12 11 16
age (mean (SD)) 39.85 (11.32) 36.50 (13.45) 51.19 (9.57) 48.85 (6.76) 0.008
GENDER (%) F 10 (50.0) 4 (33.3) 4 (36.4) 6 (37.5) 0.767
M 10 (50.0) 8 (66.7) 7 (63.6) 10 (62.5)
BMI (mean (SD)) 26.81 (6.46) 25.08 (6.73) 25.02 (3.82) 27.67 (5.95) 0.594
race (%) Asian 1 ( 5.0) 0 ( 0.0) 0 ( 0.0) 0 ( 0.0) 0.187
Black or African American 0 ( 0.0) 2 (16.7) 3 (27.3) 1 ( 6.2)
More than one race 0 ( 0.0) 1 ( 8.3) 0 ( 0.0) 0 ( 0.0)
White 19 (95.0) 9 (75.0) 8 (72.7) 15 (93.8)
HEMOGLOBIN (mean (SD)) 13.97 (1.61) 11.86 (2.59) 10.00 (2.34) 9.78 (2.39) <0.001
WBC (mean (SD)) 7.18 (1.66) 9.00 (3.34) 9.89 (6.58) 10.05 (5.12) 0.451
PLATELET (mean (SD)) 258.33 (89.63) 287.36 (146.47) 145.90 (83.98) 154.94 (88.92) 0.003
CREATININE (mean (SD)) 0.89 (0.16) 0.83 (0.41) 0.74 (0.20) 0.93 (0.54) 0.652
AST (mean (SD)) 83.33 (171.40) 28.27 (11.91) 141.70 (97.87) 109.56 (29.47) 0.036
ALT (mean (SD)) 36.89 (34.44) 21.18 (9.87) 44.30 (23.37) 49.31 (55.93) 0.308
TOTAL_BILIRUBIN (mean (SD)) 0.62 (0.49) 0.55 (0.47) 4.88 (2.60) 17.16 (9.64) <0.001
ALBUMIN (mean (SD)) 4.36 (0.24) 3.55 (0.63) 2.74 (0.51) 2.78 (0.61) <0.001
INR (mean (SD)) NaN (NA) 1.13 (0.23) 1.50 (0.41) 2.04 (0.45) <0.001
MELD_SCORE (mean (SD)) NaN (NA) 8.50 (3.50) 15.73 (4.22) 25.38 (3.93) <0.001
CHILDPUGH_SCORE (mean (SD)) NaN (NA) 5.70 (0.67) 8.70 (1.70) 10.31 (1.74) <0.001
DF_SCORE (mean (SD)) NaN (NA) -2.60 (11.66) 22.04 (20.72) 59.31 (21.56) <0.001
TLFB_AVG_drinksDD (mean (SD)) NaN (NA) 11.45 (8.30) 6.90 (3.95) 7.35 (4.21) 0.110

plasma metabolites univariate meld prediction

Significant metabolites

Primary and secondary bile acids in stool

[1] 1
[1] 2
[1] 3
[1] 4
[1] 5
[1] 6
[1] 7
[1] 8
[1] 9
[1] 10
[1] 11
[1] 12
[1] 13
[1] 14
[1] 15
[1] 16
[1] 17
[1] 18
[1] 19
[1] 20
[1] 21
[1] 22
[1] 23
[1] 24
[1] 25
[1] 26
[1] 27
[1] 28
[1] 29
[1] 30
[1] 31
[1] 32
[1] 33

Plot only those metabolites that had at least one comparison with a significant q-value.

comparison BIOCHEMICAL .y. group1 group2 n1 n2 statistic df p p.adj p.adj.signif q.value q.value.sign FoldChange log2FoldChange diffexpressed
9 AH_HDC dehydrolithocholate log10_value HDC AH 20 29 5.639 42.385 0.000 0.000 **** 0.000 TRUE 0.119 -3.070 DOWN
10 AH_HDC deoxycholate log10_value HDC AH 20 29 7.129 46.979 0.000 0.000 **** 0.000 TRUE 0.070 -3.843 DOWN
12 AH_HDC glycochenodeoxycholate glucuronide (1) log10_value HDC AH 20 29 -2.784 28.467 0.009 0.049
0.032 TRUE 14.504 3.858 UP
13 AH_HDC glycochenodeoxycholate sulfate log10_value HDC AH 20 29 -2.882 33.313 0.007 0.042
0.027 TRUE 309.187 8.272 UP
15 AH_HDC glycocholate glucuronide (1) log10_value HDC AH 20 29 -2.374 28.000 0.025 0.119 ns 0.077 TRUE 8.515 3.090 NO
16 AH_HDC glycocholate sulfate log10_value HDC AH 20 29 -2.328 28.000 0.027 0.119 ns 0.077 TRUE 24.512 4.615 NO
17 AH_HDC glycocholenate sulfate* log10_value HDC AH 20 29 -3.061 45.261 0.004 0.029
0.019 TRUE 13.998 3.807 UP
18 AH_HDC glycodeoxycholate log10_value HDC AH 20 29 6.127 23.891 0.000 0.000 **** 0.000 TRUE 0.037 -4.765 DOWN
20 AH_HDC glycolithocholate log10_value HDC AH 20 29 2.391 19.235 0.027 0.119 ns 0.077 TRUE 0.466 -1.101 NO
23 AH_HDC isoursodeoxycholate log10_value HDC AH 20 29 4.089 46.887 0.000 0.002 ** 0.001 TRUE 0.254 -1.974 DOWN
24 AH_HDC lithocholate log10_value HDC AH 20 29 7.480 41.242 0.000 0.000 **** 0.000 TRUE 0.168 -2.577 DOWN
25 AH_HDC taurochenodeoxycholate log10_value HDC AH 20 29 -3.141 46.354 0.003 0.028
0.018 TRUE 7.829 2.969 UP
27 AH_HDC taurocholenate sulfate log10_value HDC AH 20 29 -3.720 45.640 0.001 0.006 ** 0.004 TRUE 21.308 4.413 UP
28 AH_HDC taurodeoxycholate log10_value HDC AH 20 29 2.966 23.718 0.007 0.042
0.027 TRUE 0.115 -3.114 DOWN
31 AH_HDC ursocholate log10_value HDC AH 20 29 2.216 46.868 0.032 0.132 ns 0.085 TRUE 0.382 -1.388 NO
32 AH_HDC ursodeoxycholate log10_value HDC AH 20 29 2.988 46.904 0.004 0.029
0.019 TRUE 0.352 -1.506 DOWN
42 SAH_MAH dehydrolithocholate log10_value MAH SAH 8 21 0.688 13.199 0.503 0.745 ns 0.481 FALSE 2.370 1.245 NO
43 SAH_MAH deoxycholate log10_value MAH SAH 8 21 1.595 11.056 0.139 0.417 ns 0.269 FALSE 0.153 -2.709 NO
45 SAH_MAH glycochenodeoxycholate glucuronide (1) log10_value MAH SAH 8 21 -0.345 13.031 0.736 0.836 ns 0.540 FALSE 2.305 1.205 NO
46 SAH_MAH glycochenodeoxycholate sulfate log10_value MAH SAH 8 21 0.399 11.736 0.697 0.836 ns 0.540 FALSE 4.014 2.005 NO
48 SAH_MAH glycocholate glucuronide (1) log10_value MAH SAH 8 21 -0.122 14.571 0.905 0.963 ns 0.622 FALSE 2.586 1.371 NO
49 SAH_MAH glycocholate sulfate log10_value MAH SAH 8 21 0.002 10.978 0.998 0.998 ns 0.645 FALSE 1.388 0.473 NO
50 SAH_MAH glycocholenate sulfate* log10_value MAH SAH 8 21 0.332 10.153 0.747 0.836 ns 0.540 FALSE 0.324 -1.625 NO
51 SAH_MAH glycodeoxycholate log10_value MAH SAH 8 21 1.425 7.761 0.193 0.464 ns 0.300 FALSE 0.306 -1.709 NO
53 SAH_MAH glycolithocholate log10_value MAH SAH 8 21 -1.196 20.000 0.246 0.494 ns 0.319 FALSE 1.025 0.035 NO
56 SAH_MAH isoursodeoxycholate log10_value MAH SAH 8 21 1.528 11.987 0.153 0.439 ns 0.284 FALSE 0.449 -1.156 NO
57 SAH_MAH lithocholate log10_value MAH SAH 8 21 1.015 11.913 0.330 0.605 ns 0.391 FALSE 0.577 -0.793 NO
58 SAH_MAH taurochenodeoxycholate log10_value MAH SAH 8 21 -0.681 12.788 0.508 0.745 ns 0.481 FALSE 2.964 1.567 NO
60 SAH_MAH taurocholenate sulfate log10_value MAH SAH 8 21 -0.067 9.459 0.948 0.978 ns 0.632 FALSE 0.211 -2.242 NO
61 SAH_MAH taurodeoxycholate log10_value MAH SAH 8 21 0.906 8.867 0.389 0.658 ns 0.425 FALSE 0.582 -0.782 NO
64 SAH_MAH ursocholate log10_value MAH SAH 8 21 1.921 12.045 0.079 0.274 ns 0.177 FALSE 0.260 -1.945 NO
65 SAH_MAH ursodeoxycholate log10_value MAH SAH 8 21 1.365 11.010 0.200 0.464 ns 0.300 FALSE 0.361 -1.472 NO

Kruskal-Wallis tests on all metabolites to identify which are significant

ID BIOCHEMICAL KW.pvalue q.value q.value.sign Primary_Secondary Conj_Unconj Conj_type Comp12_OH Comp_12-OH_Fig1_2 Dihydroxy_Trihydroxy Comp_7-dehydroxy Comp_7-dehydro_Fig_1_2 Keto Epimer
6 M_2715 taurochenodeoxycholate 0.010 0.011 TRUE P C T n12 2 2 n7 1 n n
10 M_2727 glycocholate glucuronide (1) 0.041 0.028 TRUE P C G/Glu 12 1 2 n7 1 n n
11 M_2728 glycocholate sulfate 0.039 0.027 TRUE P C G/S 12 1 2 n7 1 n n
12 M_2729 deoxycholate 0.000 0.000 TRUE S UC 0 12 1 2 7 2 n n
13 M_2731 glycodeoxycholate 0.000 0.000 TRUE S C G 12 1 2 7 2 n n
14 M_2732 taurodeoxycholate 0.002 0.004 TRUE S C T 12 1 2 7 2 n n
15 M_2734 lithocholate 0.000 0.000 TRUE S UC 0 n12 2 1 7 2 n n
16 M_2736 glycolithocholate 0.013 0.012 TRUE S C G n12 2 1 7 2 n n
19 M_2741 ursodeoxycholate 0.010 0.011 TRUE S UC 0 n12 2 2 (1b) n7 (b) N/A n y
20 M_2742 isoursodeoxycholate 0.002 0.004 TRUE S UC 0 n12 2 2 (2b) n7 (b) N/A n y
23 M_2745 dehydrolithocholate 0.000 0.001 TRUE S UC 0 n12 2 0 7 2 y n
28 M_2760 glycocholenate sulfate* 0.033 0.024 TRUE O C G/S 12 1 2 n7 1 n n
29 M_2761 taurocholenate sulfate 0.006 0.007 TRUE O C T/S 12 1 2 n7 1 n n
32 M_2767 ursodeoxycholate sulfate (1) 0.034 0.024 TRUE S C S n12 2 1 (1b) n7 (b) N/A n y
33 M_2770 ursocholate 0.014 0.013 TRUE S UC 0 12 1 3 (2b) n7 (b) N/A n y

Primary and secondary bile acids in plasma

Note: for the glycocholate sulfate, the healthy and heavy drinking controls have the identical imputed values. Therefore one group is removed from the test. Also, be careful when comparing t-test with control

Plot only those metabolites that had at least one comparison with a significant q-value.

comparison BIOCHEMICAL .y. group1 group2 n1 n2 statistic df p p.adj p.adj.signif q.value q.value.sign FoldChange log2FoldChange diffexpressed
3 AH_HDC deoxycholate log10_value HDC AH 12 27 2.270 25.802 0.032 0.090 ns 0.086 TRUE 0.634 -0.657 DOWN
4 AH_HDC glycochenodeoxycholate log10_value HDC AH 12 27 -8.720 16.539 0.000 0.000 **** 0.000 TRUE 15.846 3.986 UP
5 AH_HDC glycochenodeoxycholate glucuronide (1) log10_value HDC AH 12 27 -6.402 21.685 0.000 0.000 **** 0.000 TRUE 8.617 3.107 UP
6 AH_HDC glycochenodeoxycholate sulfate log10_value HDC AH 12 27 -6.785 15.541 0.000 0.000 **** 0.000 TRUE 14.966 3.904 UP
7 AH_HDC glycocholate log10_value HDC AH 12 27 -10.135 20.444 0.000 0.000 **** 0.000 TRUE 17.249 4.108 UP
8 AH_HDC glycocholate sulfate log10_value HDC AH 12 27 -6.106 26.000 0.000 0.000 **** 0.000 TRUE 15.651 3.968 UP
9 AH_HDC glycocholenate sulfate* log10_value HDC AH 12 27 -4.551 21.249 0.000 0.001 *** 0.001 TRUE 3.258 1.704 UP
10 AH_HDC glycodeoxycholate log10_value HDC AH 12 27 2.849 25.443 0.009 0.034
0.033 TRUE 3.053 1.610 UP
12 AH_HDC glycolithocholate log10_value HDC AH 12 27 -2.567 36.993 0.014 0.048
0.046 TRUE 15.606 3.964 UP
13 AH_HDC glycolithocholate sulfate* log10_value HDC AH 12 27 -2.884 13.729 0.012 0.043
0.041 TRUE 4.061 2.022 UP
14 AH_HDC glycoursodeoxycholate log10_value HDC AH 12 27 -2.417 24.874 0.023 0.075 ns 0.071 TRUE 8.129 3.023 UP
15 AH_HDC isoursodeoxycholate log10_value HDC AH 12 27 3.736 18.583 0.001 0.005 ** 0.004 TRUE 0.174 -2.520 DOWN
16 AH_HDC taurochenodeoxycholate log10_value HDC AH 12 27 -12.057 14.596 0.000 0.000 **** 0.000 TRUE 93.188 6.542 UP
17 AH_HDC taurocholate log10_value HDC AH 12 27 -9.910 19.373 0.000 0.000 **** 0.000 TRUE 127.817 6.998 UP
18 AH_HDC taurocholenate sulfate log10_value HDC AH 12 27 -7.315 14.536 0.000 0.000 **** 0.000 TRUE 11.732 3.552 UP
19 AH_HDC taurodeoxycholate log10_value HDC AH 12 27 2.823 24.406 0.009 0.034
0.033 TRUE 5.281 2.401 UP
20 AH_HDC taurolithocholate 3-sulfate log10_value HDC AH 12 27 -5.973 14.333 0.000 0.000 *** 0.000 TRUE 15.564 3.960 UP
21 AH_HDC tauroursodeoxycholate log10_value HDC AH 12 27 -6.853 19.087 0.000 0.000 **** 0.000 TRUE 130.767 7.031 UP
25 HDC_HC deoxycholate log10_value HC HDC 20 12 2.123 21.283 0.046 0.115 ns 0.110 FALSE 0.587 -0.769 NO
26 HDC_HC glycochenodeoxycholate log10_value HC HDC 20 12 -3.774 20.920 0.001 0.005 ** 0.004 TRUE 4.247 2.087 UP
27 HDC_HC glycochenodeoxycholate glucuronide (1) log10_value HC HDC 20 12 -1.703 26.319 0.100 0.224 ns 0.214 FALSE 1.526 0.610 NO
28 HDC_HC glycochenodeoxycholate sulfate log10_value HC HDC 20 12 -3.132 19.137 0.005 0.022
0.021 TRUE 6.320 2.660 UP
29 HDC_HC glycocholate log10_value HC HDC 20 12 -4.637 27.874 0.000 0.000 *** 0.000 TRUE 3.110 1.637 UP
30 HDC_HC glycocholenate sulfate* log10_value HC HDC 20 12 -1.891 15.506 0.078 0.181 ns 0.173 FALSE 1.741 0.800 NO
31 HDC_HC glycodeoxycholate log10_value HC HDC 20 12 -0.041 18.315 0.968 0.985 ns 0.940 FALSE 1.293 0.370 NO
33 HDC_HC glycolithocholate log10_value HC HDC 20 12 0.329 25.225 0.745 0.866 ns 0.826 FALSE 0.827 -0.274 NO
34 HDC_HC glycolithocholate sulfate* log10_value HC HDC 20 12 0.730 12.246 0.479 0.708 ns 0.675 FALSE 1.642 0.716 NO
35 HDC_HC glycoursodeoxycholate log10_value HC HDC 20 12 -2.311 20.022 0.032 0.090 ns 0.086 TRUE 3.007 1.588 UP
36 HDC_HC isoursodeoxycholate log10_value HC HDC 20 12 1.558 21.088 0.134 0.272 ns 0.260 FALSE 0.369 -1.437 NO
37 HDC_HC taurochenodeoxycholate log10_value HC HDC 20 12 -2.238 19.338 0.037 0.096 ns 0.092 TRUE 2.823 1.497 UP
38 HDC_HC taurocholate log10_value HC HDC 20 12 -2.331 22.045 0.029 0.090 ns 0.086 TRUE 2.911 1.541 UP
39 HDC_HC taurocholenate sulfate log10_value HC HDC 20 12 -0.007 16.079 0.994 0.994 ns 0.948 FALSE 1.812 0.858 NO
40 HDC_HC taurodeoxycholate log10_value HC HDC 20 12 0.378 16.953 0.710 0.866 ns 0.826 FALSE 1.269 0.344 NO
41 HDC_HC taurolithocholate 3-sulfate log10_value HC HDC 20 12 0.830 14.507 0.420 0.702 ns 0.670 FALSE 1.571 0.652 NO
42 HDC_HC tauroursodeoxycholate log10_value HC HDC 20 12 -2.319 15.173 0.035 0.095 ns 0.090 TRUE 5.382 2.428 UP
46 SAH_MAH deoxycholate log10_value MAH SAH 11 16 0.745 15.237 0.468 0.708 ns 0.675 FALSE 0.376 -1.410 NO
47 SAH_MAH glycochenodeoxycholate log10_value MAH SAH 11 16 0.163 19.600 0.872 0.977 ns 0.932 FALSE 0.957 -0.064 NO
48 SAH_MAH glycochenodeoxycholate glucuronide (1) log10_value MAH SAH 11 16 0.050 18.412 0.961 0.985 ns 0.940 FALSE 0.708 -0.499 NO
49 SAH_MAH glycochenodeoxycholate sulfate log10_value MAH SAH 11 16 0.464 20.345 0.648 0.826 ns 0.788 FALSE 0.762 -0.392 NO
50 SAH_MAH glycocholate log10_value MAH SAH 11 16 0.552 16.172 0.588 0.764 ns 0.729 FALSE 0.712 -0.490 NO
51 SAH_MAH glycocholate sulfate log10_value MAH SAH 11 16 0.651 20.915 0.522 0.719 ns 0.686 FALSE 0.525 -0.930 NO
52 SAH_MAH glycocholenate sulfate* log10_value MAH SAH 11 16 0.677 23.025 0.505 0.714 ns 0.681 FALSE 0.963 -0.055 NO
53 SAH_MAH glycodeoxycholate log10_value MAH SAH 11 16 1.487 10.000 0.168 0.321 ns 0.306 FALSE 0.011 -6.519 NO
55 SAH_MAH glycolithocholate log10_value MAH SAH 11 16 -0.233 15.541 0.819 0.934 ns 0.891 FALSE 0.371 -1.431 NO
56 SAH_MAH glycolithocholate sulfate* log10_value MAH SAH 11 16 0.095 13.664 0.926 0.985 ns 0.940 FALSE 0.397 -1.333 NO
57 SAH_MAH glycoursodeoxycholate log10_value MAH SAH 11 16 0.637 22.843 0.531 0.719 ns 0.686 FALSE 4.006 2.002 NO
58 SAH_MAH isoursodeoxycholate log10_value MAH SAH 11 16 1.935 21.252 0.066 0.159 ns 0.152 FALSE 0.502 -0.993 NO
59 SAH_MAH taurochenodeoxycholate log10_value MAH SAH 11 16 -1.666 16.862 0.114 0.239 ns 0.228 FALSE 1.589 0.668 NO
60 SAH_MAH taurocholate log10_value MAH SAH 11 16 -0.806 21.233 0.429 0.702 ns 0.670 FALSE 1.629 0.704 NO
61 SAH_MAH taurocholenate sulfate log10_value MAH SAH 11 16 -0.131 23.542 0.897 0.985 ns 0.940 FALSE 1.419 0.505 NO
62 SAH_MAH taurodeoxycholate log10_value MAH SAH 11 16 1.491 10.000 0.167 0.321 ns 0.306 FALSE 0.003 -8.582 NO
63 SAH_MAH taurolithocholate 3-sulfate log10_value MAH SAH 11 16 -0.739 17.094 0.470 0.708 ns 0.675 FALSE 0.901 -0.150 NO
64 SAH_MAH tauroursodeoxycholate log10_value MAH SAH 11 16 -0.683 23.997 0.501 0.714 ns 0.681 FALSE 9.394 3.232 NO

Correlation of significant BA with taxa

HDC and AH correlations

MAH and SAH correlations

SAH correlations

Taxa sensitive and resistant to bile acids

Taxa linked to bile acids

Conjugation group heatmaps

Stool total primary and total secondary heatmaps

Boxplots of the total primary concentration in each sample

T-tests on the log scale: primary

total.primary.lm_stool <- lm(log10(value) ~ Class, data = subset(total.primary_stool, 
    Class != "AH" & Primary_Secondary == "P"))
anova(total.primary.lm_stool)
Analysis of Variance Table

Response: log10(value)
          Df  Sum Sq Mean Sq F value Pr(>F)
Class      2  0.9575 0.47874  1.0076  0.373
Residuals 46 21.8554 0.47512               
total.primary.lsm <- lsmeans::lsmeans(total.primary.lm_stool, pairwise ~ Class)
total.primary.lsm
$lsmeans
 Class lsmean    SE df lower.CL upper.CL
 HDC     1.06 0.154 46    0.751     1.37
 MAH     1.38 0.244 46    0.887     1.87
 SAH     1.33 0.150 46    1.028     1.63

Results are given on the log10 (not the response) scale. 
Confidence level used: 0.95 

$contrasts
 contrast  estimate    SE df t.ratio p.value
 HDC - MAH  -0.3164 0.288 46 -1.097  0.5205 
 HDC - SAH  -0.2696 0.215 46 -1.252  0.4294 
 MAH - SAH   0.0469 0.286 46  0.164  0.9854 

Note: contrasts are still on the log10 scale 
P value adjustment: tukey method for comparing a family of 3 estimates 

T-tests on the log scale: secondary

total.secondary.lm_stool <- lm(log10(value) ~ Class, data = subset(total.primary_stool, 
    Class != "AH" & Primary_Secondary == "S"))
anova(total.secondary.lm_stool)
Analysis of Variance Table

Response: log10(value)
          Df Sum Sq Mean Sq F value Pr(>F)  
Class      2  2.750 1.37498  4.2642   0.02 *
Residuals 46 14.833 0.32245                 
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
total.secondary.lsm <- lsmeans::lsmeans(total.secondary.lm_stool, pairwise ~ Class)
total.secondary.lsm
$lsmeans
 Class lsmean    SE df lower.CL upper.CL
 HDC     1.72 0.127 46    1.460     1.97
 MAH     1.61 0.201 46    1.211     2.02
 SAH     1.21 0.124 46    0.964     1.46

Results are given on the log10 (not the response) scale. 
Confidence level used: 0.95 

$contrasts
 contrast  estimate    SE df t.ratio p.value
 HDC - MAH    0.101 0.238 46 0.424   0.9057 
 HDC - SAH    0.502 0.177 46 2.832   0.0184 
 MAH - SAH    0.402 0.236 46 1.702   0.2153 

Note: contrasts are still on the log10 scale 
P value adjustment: tukey method for comparing a family of 3 estimates 

Conjugated unconjugated

Boxplots of the total conjugated concentration in each sample

T-tests on the log scale: conjugated

total.conj.lm_stool <- lm(log10(value) ~ Class, data = subset(total.conj_stool, Class != 
    "AH" & Conj_Unconj == "C"))
anova(total.conj.lm_stool)
Analysis of Variance Table

Response: log10(value)
          Df  Sum Sq Mean Sq F value Pr(>F)
Class      2  0.3214 0.16068  0.3541 0.7037
Residuals 46 20.8722 0.45374               

T-tests on the log scale: unconjugated

total.unconj.lm_stool <- lm(log10(value) ~ Class, data = subset(total.conj_stool, 
    Class != "AH" & Conj_Unconj == "UC"))
anova(total.unconj.lm_stool)
Analysis of Variance Table

Response: log10(value)
          Df  Sum Sq Mean Sq F value  Pr(>F)  
Class      2  2.6863 1.34313   2.618 0.08379 .
Residuals 46 23.5998 0.51304                  
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
total.unconj.lsm <- lsmeans::lsmeans(total.unconj.lm_stool, pairwise ~ Class)
total.unconj.lsm
$lsmeans
 Class lsmean    SE df lower.CL upper.CL
 HDC    1.496 0.160 46    1.174     1.82
 MAH    1.304 0.253 46    0.795     1.81
 SAH    0.987 0.156 46    0.672     1.30

Results are given on the log10 (not the response) scale. 
Confidence level used: 0.95 

$contrasts
 contrast  estimate    SE df t.ratio p.value
 HDC - MAH    0.192 0.300 46 0.640   0.7991 
 HDC - SAH    0.509 0.224 46 2.275   0.0696 
 MAH - SAH    0.317 0.298 46 1.067   0.5394 

Note: contrasts are still on the log10 scale 
P value adjustment: tukey method for comparing a family of 3 estimates 

Plasma total primary and total secondary heatmaps

Boxplots of the total secondary concentration in each sample

T-tests on the log scale:primary

total.primary.lm_plasma <- lm(log10(value) ~ Class, data = subset(total.primary_plasma, 
    Class != "AH" & Primary_Secondary == "P"))
anova(total.primary.lm_plasma)
Analysis of Variance Table

Response: log10(value)
          Df  Sum Sq Mean Sq F value    Pr(>F)    
Class      3 28.9838  9.6613  81.042 < 2.2e-16 ***
Residuals 55  6.5567  0.1192                      
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
total.primary.lsm <- lsmeans::lsmeans(total.primary.lm_plasma, pairwise ~ Class)
total.primary.lsm
$lsmeans
 Class lsmean     SE df lower.CL upper.CL
 HC     0.505 0.0772 55    0.350    0.660
 HDC    0.721 0.0997 55    0.521    0.921
 MAH    1.918 0.1041 55    1.709    2.127
 SAH    2.026 0.0863 55    1.854    2.199

Results are given on the log10 (not the response) scale. 
Confidence level used: 0.95 

$contrasts
 contrast  estimate    SE df t.ratio p.value
 HC - HDC    -0.216 0.126 55  -1.715 0.3259 
 HC - MAH    -1.413 0.130 55 -10.904 <.0001 
 HC - SAH    -1.522 0.116 55 -13.140 <.0001 
 HDC - MAH   -1.197 0.144 55  -8.306 <.0001 
 HDC - SAH   -1.305 0.132 55  -9.901 <.0001 
 MAH - SAH   -0.108 0.135 55  -0.801 0.8534 

Note: contrasts are still on the log10 scale 
P value adjustment: tukey method for comparing a family of 4 estimates 

T-tests on the log scale: secondary

total.secondary.lm_plasma <- lm(log10(value) ~ Class, data = subset(total.primary_plasma, 
    Class != "AH" & Primary_Secondary == "S"))
anova(total.secondary.lm_plasma)
Analysis of Variance Table

Response: log10(value)
          Df  Sum Sq Mean Sq F value Pr(>F)
Class      3  1.2743 0.42475  1.7944  0.159
Residuals 55 13.0188 0.23671               
total.secondary.lsm <- lsmeans::lsmeans(total.secondary.lm_plasma, pairwise ~ Class)
total.secondary.lsm
$lsmeans
 Class lsmean    SE df lower.CL upper.CL
 HC      1.11 0.109 55    0.893     1.33
 HDC     1.05 0.140 55    0.766     1.33
 MAH     1.41 0.147 55    1.116     1.70
 SAH     1.35 0.122 55    1.109     1.60

Results are given on the log10 (not the response) scale. 
Confidence level used: 0.95 

$contrasts
 contrast  estimate    SE df t.ratio p.value
 HC - HDC    0.0636 0.178 55  0.358  0.9841 
 HC - MAH   -0.2992 0.183 55 -1.638  0.3661 
 HC - SAH   -0.2416 0.163 55 -1.481  0.4560 
 HDC - MAH  -0.3628 0.203 55 -1.786  0.2908 
 HDC - SAH  -0.3052 0.186 55 -1.643  0.3638 
 MAH - SAH   0.0576 0.191 55  0.302  0.9903 

Note: contrasts are still on the log10 scale 
P value adjustment: tukey method for comparing a family of 4 estimates 

Conjugated unconjugated

Boxplots of the total conjugated concentration in each sample

T-tests on the log scale: conjugated

# Conj_Unconj == 'C'
total.conj.lm_plasma <- lm(log10(value) ~ Class, data = subset(total.conj_plasma, 
    Class != "AH" & Conj_Unconj == "C"))
anova(total.conj.lm_plasma)
Analysis of Variance Table

Response: log10(value)
          Df  Sum Sq Mean Sq F value    Pr(>F)    
Class      3 23.3016  7.7672   80.08 < 2.2e-16 ***
Residuals 55  5.3346  0.0970                      
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
total.conj.lsm <- lsmeans::lsmeans(total.conj.lm_plasma, pairwise ~ Class)
total.conj.lsm
$lsmeans
 Class lsmean     SE df lower.CL upper.CL
 HC     0.815 0.0696 55    0.675    0.954
 HDC    1.016 0.0899 55    0.835    1.196
 MAH    2.123 0.0939 55    1.935    2.311
 SAH    2.157 0.0779 55    2.001    2.313

Results are given on the log10 (not the response) scale. 
Confidence level used: 0.95 

$contrasts
 contrast  estimate    SE df t.ratio p.value
 HC - HDC    -0.201 0.114 55  -1.764 0.3013 
 HC - MAH    -1.308 0.117 55 -11.189 <.0001 
 HC - SAH    -1.342 0.104 55 -12.848 <.0001 
 HDC - MAH   -1.107 0.130 55  -8.519 <.0001 
 HDC - SAH   -1.141 0.119 55  -9.598 <.0001 
 MAH - SAH   -0.034 0.122 55  -0.279 0.9924 

Note: contrasts are still on the log10 scale 
P value adjustment: tukey method for comparing a family of 4 estimates 

T-tests on the log scale: unconjugated

total.unconj.lm_plasma <- lm(log10(value) ~ Class, data = subset(total.conj_plasma, 
    Class != "AH" & Conj_Unconj == "UC"))
anova(total.unconj.lm_plasma)
Analysis of Variance Table

Response: log10(value)
          Df  Sum Sq Mean Sq F value Pr(>F)
Class      3  0.5114 0.17047  0.7029 0.5544
Residuals 55 13.3401 0.24255               

Precursor: 7-alpha-hydroxy-3-oxo-4-cholestenoate (7-Hoca) 2530

T-tests on the log scale:7-HOCA

HOCA.lm_plasma <- lm(log10(value) ~ Class, data = M_2530_df.m)
anova(HOCA.lm_plasma)
Analysis of Variance Table

Response: log10(value)
          Df Sum Sq Mean Sq F value    Pr(>F)    
Class      3 4.0157 1.33856  21.627 2.214e-09 ***
Residuals 55 3.4041 0.06189                      
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
HOCA.lm_plasma.lsm <- lsmeans::lsmeans(HOCA.lm_plasma, pairwise ~ Class)
HOCA.lm_plasma.lsm
$lsmeans
 Class  lsmean     SE df lower.CL upper.CL
 HC    -0.0394 0.0556 55   -0.151   0.0721
 HDC   -0.2093 0.0718 55   -0.353  -0.0654
 MAH    0.2807 0.0750 55    0.130   0.4310
 SAH    0.4667 0.0622 55    0.342   0.5913

Results are given on the log10 (not the response) scale. 
Confidence level used: 0.95 

$contrasts
 contrast  estimate     SE df t.ratio p.value
 HC - HDC     0.170 0.0908 55  1.871  0.2525 
 HC - MAH    -0.320 0.0934 55 -3.428  0.0062 
 HC - SAH    -0.506 0.0834 55 -6.064  <.0001 
 HDC - MAH   -0.490 0.1038 55 -4.719  0.0001 
 HDC - SAH   -0.676 0.0950 55 -7.115  <.0001 
 MAH - SAH   -0.186 0.0974 55 -1.908  0.2365 

Note: contrasts are still on the log10 scale 
P value adjustment: tukey method for comparing a family of 4 estimates 

Figure 1

put 7-hoca in the middel as separator

Figure 2: significant metabolites in healthy to heavy drinking comparison

Figure 5: Correlation plots

Stool Random forest models for prediction of AH and disease severity

AH prediction model in stool


Call:
 randomForest(x = x, y = y, ntree = 501, mtry = param$mtry, importance = TRUE) 
               Type of random forest: classification
                     Number of trees: 501
No. of variables tried at each split: 5

        OOB estimate of  error rate: 14.29%
Confusion matrix:
    AH HDC class.error
AH  25   4    0.137931
HDC  3  17    0.150000

Disease severity in stool


Call:
 randomForest(x = x, y = y, ntree = 501, mtry = param$mtry, importance = TRUE) 
               Type of random forest: classification
                     Number of trees: 501
No. of variables tried at each split: 6

        OOB estimate of  error rate: 34.48%
Confusion matrix:
    MAH SAH class.error
MAH   1   7   0.8750000
SAH   3  18   0.1428571

Plasma Random forest models for prediction of AH and disease severity

AH prediction model in plasma


Call:
 randomForest(x = x, y = y, ntree = 501, mtry = param$mtry, importance = TRUE) 
               Type of random forest: classification
                     Number of trees: 501
No. of variables tried at each split: 4

        OOB estimate of  error rate: 2.56%
Confusion matrix:
    AH HDC class.error
AH  26   1  0.03703704
HDC  0  12  0.00000000

Disease severity in plasma


Call:
 randomForest(x = x, y = y, ntree = 501, mtry = param$mtry, importance = TRUE) 
               Type of random forest: classification
                     Number of trees: 501
No. of variables tried at each split: 5

        OOB estimate of  error rate: 44.44%
Confusion matrix:
    MAH SAH class.error
MAH   3   8   0.7272727
SAH   4  12   0.2500000

Figure 4

Supplementary figure 1

Prediction of disease severity

MELD score univariate prediction

Stool metabolites univariate meld prediction

BIOCHEMICAL SUPER_PATHWAY SUB_PATHWAY coeff_AH p_val_AH R_sq_AH coeff_Sev p_val_Sev R_sq_Sev
cholate Lipid Primary Bile Acid Metabolism -1.400 0.359 0.018 -1.947 0.238 0.051
glycocholate Lipid Primary Bile Acid Metabolism -0.101 0.948 0.000 -0.413 0.765 0.003
taurocholate Lipid Primary Bile Acid Metabolism 1.177 0.451 0.012 0.460 0.738 0.004
chenodeoxycholate Lipid Primary Bile Acid Metabolism 0.244 0.875 0.001 -0.732 0.631 0.009
glycochenodeoxycholate Lipid Primary Bile Acid Metabolism 1.265 0.460 0.012 -0.086 0.958 0.000
taurochenodeoxycholate Lipid Primary Bile Acid Metabolism 4.498 0.006 0.151 1.654 0.320 0.037
cholate sulfate Lipid Primary Bile Acid Metabolism -3.895 0.083 0.064 -2.937 0.242 0.050
glycochenodeoxycholate glucuronide (1) Lipid Primary Bile Acid Metabolism 4.613 0.112 0.054 -0.471 0.842 0.002
glycochenodeoxycholate sulfate Lipid Primary Bile Acid Metabolism 1.821 0.244 0.029 -1.110 0.388 0.028
glycocholate glucuronide (1) Lipid Primary Bile Acid Metabolism 3.394 0.321 0.021 -1.833 0.498 0.017
glycocholate sulfate Lipid Primary Bile Acid Metabolism 2.763 0.315 0.022 -1.348 0.534 0.014
deoxycholate Lipid Secondary Bile Acid Metabolism -3.619 0.000 0.393 -0.851 0.402 0.026
glycodeoxycholate Lipid Secondary Bile Acid Metabolism -8.304 0.000 0.429 -6.982 0.102 0.096
taurodeoxycholate Lipid Secondary Bile Acid Metabolism -7.870 0.004 0.167 -1.701 0.748 0.004
lithocholate Lipid Secondary Bile Acid Metabolism -4.154 0.000 0.384 -0.954 0.384 0.028
glycolithocholate Lipid Secondary Bile Acid Metabolism -14.981 0.021 0.110 3.238 0.947 0.000
glycolithocholate sulfate* Lipid Secondary Bile Acid Metabolism -1.496 0.410 0.015 -2.312 0.165 0.070
taurolithocholate 3-sulfate Lipid Secondary Bile Acid Metabolism 0.066 0.976 0.000 -2.273 0.227 0.054
ursodeoxycholate Lipid Secondary Bile Acid Metabolism -3.690 0.005 0.158 -1.166 0.370 0.030
isoursodeoxycholate Lipid Secondary Bile Acid Metabolism -5.623 0.001 0.230 -1.689 0.331 0.035
glycoursodeoxycholate Lipid Secondary Bile Acid Metabolism -4.369 0.030 0.099 -3.600 0.073 0.114
tauroursodeoxycholate Lipid Secondary Bile Acid Metabolism -1.014 0.754 0.002 -2.739 0.357 0.032
dehydrolithocholate Lipid Secondary Bile Acid Metabolism -3.969 0.000 0.314 -1.056 0.414 0.025
7,12-diketolithocholate Lipid Secondary Bile Acid Metabolism -1.915 0.542 0.008 1.589 0.655 0.008
7-ketolithocholate Lipid Secondary Bile Acid Metabolism -0.083 0.952 0.000 -1.266 0.384 0.028
3-dehydrocholate Lipid Secondary Bile Acid Metabolism 0.462 0.772 0.002 -0.078 0.965 0.000
12-dehydrocholate Lipid Secondary Bile Acid Metabolism -0.847 0.586 0.006 -0.013 0.994 0.000
glycocholenate sulfate* Lipid Secondary Bile Acid Metabolism 2.294 0.171 0.040 -1.360 0.365 0.031
taurocholenate sulfate Lipid Secondary Bile Acid Metabolism 3.143 0.034 0.095 -0.535 0.703 0.005
7-ketodeoxycholate Lipid Secondary Bile Acid Metabolism -1.911 0.218 0.033 -1.736 0.304 0.039
glycodeoxycholate sulfate Lipid Secondary Bile Acid Metabolism -2.873 0.265 0.027 -2.686 0.227 0.054
ursodeoxycholate sulfate (1) Lipid Secondary Bile Acid Metabolism -4.304 0.002 0.197 -3.670 0.049 0.136
ursocholate Lipid Secondary Bile Acid Metabolism -3.220 0.014 0.125 -1.397 0.257 0.047

Severity prediction model with significant metabolite

Plasma meld prediction

plasma metabolites univariate meld prediction

BIOCHEMICAL SUPER_PATHWAY SUB_PATHWAY coeff_AH p_val_AH R_sq_AH coeff_Sev p_val_Sev R_sq_Sev
cholate Lipid Primary Bile Acid Metabolism 2.032 0.440 0.017 2.679 0.228 0.058
glycocholate Lipid Primary Bile Acid Metabolism 8.663 0.000 0.473 3.293 0.365 0.033
taurocholate Lipid Primary Bile Acid Metabolism 4.882 0.000 0.504 3.041 0.141 0.084
chenodeoxycholate Lipid Primary Bile Acid Metabolism 4.018 0.169 0.053 3.171 0.256 0.051
glycochenodeoxycholate Lipid Primary Bile Acid Metabolism 7.873 0.000 0.416 2.820 0.453 0.023
taurochenodeoxycholate Lipid Primary Bile Acid Metabolism 5.625 0.000 0.552 7.968 0.022 0.192
glycochenodeoxycholate glucuronide (1) Lipid Primary Bile Acid Metabolism 8.361 0.000 0.349 3.474 0.264 0.050
glycochenodeoxycholate sulfate Lipid Primary Bile Acid Metabolism 6.961 0.000 0.437 4.179 0.156 0.079
glycocholate sulfate Lipid Primary Bile Acid Metabolism 5.699 0.006 0.198 1.134 0.570 0.013
deoxycholate Lipid Secondary Bile Acid Metabolism -4.306 0.132 0.064 -0.281 0.913 0.000
glycodeoxycholate Lipid Secondary Bile Acid Metabolism -4.256 0.020 0.144 -2.093 0.230 0.057
taurodeoxycholate Lipid Secondary Bile Acid Metabolism -3.312 0.024 0.137 -1.767 0.207 0.063
glycolithocholate Lipid Secondary Bile Acid Metabolism 2.895 0.094 0.078 0.813 0.582 0.012
glycolithocholate sulfate* Lipid Secondary Bile Acid Metabolism 5.021 0.005 0.206 1.227 0.652 0.008
taurolithocholate 3-sulfate Lipid Secondary Bile Acid Metabolism 5.777 0.000 0.439 3.726 0.160 0.077
ursodeoxycholate Lipid Secondary Bile Acid Metabolism -0.280 0.865 0.001 1.719 0.240 0.055
isoursodeoxycholate Lipid Secondary Bile Acid Metabolism -6.620 0.000 0.299 -2.510 0.284 0.046
glycoursodeoxycholate Lipid Secondary Bile Acid Metabolism 3.747 0.054 0.102 2.148 0.237 0.056
tauroursodeoxycholate Lipid Secondary Bile Acid Metabolism 5.633 0.000 0.488 4.032 0.030 0.174
glycocholenate sulfate* Lipid Secondary Bile Acid Metabolism 10.060 0.004 0.219 0.101 0.981 0.000
taurocholenate sulfate Lipid Secondary Bile Acid Metabolism 7.321 0.000 0.472 3.683 0.306 0.042
glycodeoxycholate sulfate Lipid Secondary Bile Acid Metabolism -0.814 0.691 0.005 -1.190 0.491 0.019

Severity prediction model with significant metabolite

Figure 3: significant metabolites in M to S alcoholic hepatitis comparison